Mitochondrial matrix Ca2+ efflux is largely mediated by the mitochondrial Na+/Ca2+ exchanger (NCLX), directly coupling mitochondrial Ca2+ and Na+ handling. NCLX has been shown to play a central role in many pathophysiological processes, including colorectal tumor metastasis, and Parkinson’s and Alzheimer diseases. Indeed, sporadic Alzheimer´s disease patients had decreased frontal cortex NCLX levels, an effect recapitulated in mouse models. However, transcriptional mechanisms specifying its functional states has not been clearly defined. Here, we aimed to investigate how NCLX expression is regulated. NCLX mRNA was consistently found to be increased after mild stressful events, including scrambled siRNA transfection and control adenoviral vector transduction. We then hypothesized that NCLX gene expression was controlled by a stress‐sensitive pathway. Indeed, serum‐deprived astrocytes and mouse embryonic fibroblasts (MEFs) showed an increase in NCLX mRNA levels, an effect that was even further modulated by incubation with thapsigargin or H2O2, indicating expression sensitivity to a wide variety of stress signals. Here, we took advantage of a genome‐wide approach using available epigenomics datasets, and transcriptomic data to pinpoint transcriptional factors that orchestrate NCLX gene expression upon homeostatic conditions in astrocytes. We identified promoter‐distal enhancer‐like elements in forebrain mouse and primary astrocytes using chromatin immunoprecipitation high‐throughput sequencing (ChIP‐seq) data marked by histone acetylation of lysine 27 (H3K27ac). We also used a public ATAC‐seq to define a coincident region with accessible chromatin. Still, transcription factor binding sites uncovered through sequence‐based single site analysis of the NCLX gene indicated several putative transcription factors, including many components of the NF‐κB family. In parallel, public RNA‐seq databases were mined for patterns that were correlated with transcription factor known effects, before proceeding with binding confirmation through ChIP‐PCR. In summary, we were able to build a foundation to test the modulation of NCLX expression. This proof‐of‐concept of NCLX stress‐induced mechanism may be further explored as a tool for targeting cellular Na+/Ca2+signaling, mitochondrial function, and metabolism.
Autophagy is an essential cellular process regulated by intracellular calcium signals, which play an important role in autophagic activation during metabolic changes. Calcium transporters are present in mitochondria, an organelle able to uptake and release calcium ions, thus participating in cellular calcium signaling. Uptake by mitochondria is mediated by the mitochondrial calcium uniporter (MCU), while extrusion occurs through the mitochondrial sodium/lithium/calcium exchanger (NCLX). The aim of this work was to investigate how MCU and NCLX affect autophagy. To do so, we evaluated makers of autophagic activity in murine Aml‐12 hepatic cells transfected with siRNAs targeting either MCU or NCLX expression, leading to genetic knockdown (KD). Additionally, we investigated the autophagic response to serum/amino acid starvation and rapamycin (mTORC1 inhibitor) treatment in Aml‐12 cells with NCLX KD or pharmacological inhibition by CGP37157 (CGP). Using a cell line stably expressing the autophagic probe LC3‐GFP‐mCherry, we observed that NCLX KD leads to impaired autophagosome formation under basal conditions. Curiously, this effect was associated with a significant decrease in mRNA expression of LC3A and LC3B genes, while the expression of other autophagy‐related genes, such as TFEB, ATG5, ATG12, and ATG7, was upregulated or unchanged. Conversely, MCU KD led to an apparent increase in autophagosome and autolysosome numbers, indicating enhanced autophagic activity. Interestingly, MCU KD also led to decreased expression of LC3A, but not LC3B. The expression of TFEB, ATG12, ATG5, and ATG7 were decreased or unchanged by MCU KD. After autophagic stimulation by serum/amino acid starvation, the levels of LC3 II were lower in NCLX KD cells compared to negative control in the presence or absence of bafilomycin A1, which indicates a reduction of autophagic flux. The levels of LC3 I were significantly lower in NCLX KD cells under basal and stimulated conditions, corroborating the decreased LC3 mRNA levels observed. Importantly, these effects were also observed using CGP to inhibit NCLX. The reduction of autophagic flux by NCLX KD or CGP was not observed in cells treated with rapamycin. We also measured the levels of phosphorylated 4E‐BP1 as an indication of mTORC1 activity. As expected, serum/amino acid starvation and rapamycin decreased the levels of p‐4E‐BP1; however, this decrease was modulated by CGP only in starved cells, indicating that NCLX may affect mTORC1 activity in an upstream pathway. In conclusion, we show that mitochondrial calcium transporters are novel autophagy‐regulating pathways: MCU modulates autophagic activation under basal conditions, while NCLX maintains autophagic activity under basal and stimulated conditions.
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